Project Summary/Abstract The aim of this proposal is to identify and characterize novel long intergenic noncoding RNAs (lincRNAs) that function in NF?B signaling. Macrophages are critical effector cells of the innate immune system where they function to control infection and maintain tissue homeostasis. Activation of the innate immune response occurs through germline encoded receptors such as the Toll like receptors that act as pathogen recognition receptors activating inflammation. This is a tightly regulated procedure and here we aim to characterize the role that lincRNAs play in controlling the activation of NF?B downstream of TLR stimulation. We will carry out a systematic study of 18 lincRNAs that we have identified using RNA-sequencing as the most highly inducible lincRNAs following TLR activation. We will make use of our recently developed murine NF?B/Cas9 reporter macrophage cell line to allow rapid progress in screening for lincRNAs critical in this response. We have developed a dual guide RNA system that will enable us to knockout all 18 lincRNAs and identify those that are critical in controlling NF?B activity. This will allow for rapid meaningful data to be obtained in a highly efficient manner. We will characterize the possible mechanism of action of these lincRNAs in controlling inflammation by carrying out RNA-seq on deficient cells following inflammatory stimulation. We have begun to establish our mechanistic analysis pipeline by initially characterizing the functions of one lincRNA named lincRNA-Cox2. Cells deficient in lincRNA- Cox2 using the dual guide RNA method results in ~60% decrease in NF?B activity and functions to control specific sets of innate immune genes. This project will establish a foundation for future studies to determine the regulatory importance of lincRNAs in inflammatory diseases.